1. Field of the Invention
The present invention relates to a new simple method for determining human prolyl hydroxylase, which is useful in the straightforward diagnosis of hepatic diseases. More particularly, the present invention relates to a method for determining human prolyl hydroxylase by radioimmunoassay according to the sandwich method using a specific monoclonal antibody to human prolyl hydroxylase as at least one of the antibodies which are to be coated on a solid phase (support) and to be labeled with a radioactive element.
2. Description of the Prior Art
Among methods known hitherto for determining human prolyl hydroxylase (referred to hereinafter simply as hPH) in human blood is included a method wherein 4-L-proline in protocollagen labeled with .sup.3 H is used as a substrate and the resultant .sup.3 H labeled water is captured by vacuum distillation and measured for its radioactivity (Hutton et al., Anal. Biochem., 16, 384-394, 1966). Other known methods involve the use of .sup.14 C-proline labeled protocollagen as a substrate followed by the measurement of radioactivity from the resultant 4-hydroxylated .sup.14 C-proline (Juva et al., Anal. Biochem. 15, 77-83, 1966); or the use of (pro-pro-gly).sub.5 or (pro-pro-gly).sub.10 as a substrate followed by the capture and measurement of .sup.14 CO.sub.2 released from 2-oxo (1-.sup.14 C)-glutaric acid (Berg et al., J. Biol, Chem., 248, 1175-1182, 1973). Any of these methods, however, has disadvantages of requiring complicated, tremendous operations and time-consuming measurements. Furthermore, a simple measurement of hPH activity in blood does not reflect the true hPH level, because most of the hPH is present in blood in an enzymologically inactivated state.
As an immunoassay is known a radioimmunoassay described by Tuderman et al. in Bur. J. Biochem., 60, 399-405, 1975, wherein a labeled antigen is used, utilizing a competitive reaction between the labeled and unlabeled antigens, and determination of hPH is effected after the subsequent reaction with a second antibody. The antibody employed in this competitive reaction is a rabbit polyclonal antibody to hPH. Thus, immunoassay results in low specificity and shows a sensitivity of 5-10 ng for hPH. Moreover, this method is not simple to operate, e.g. requiring centrifugation steps for the separation of the antigen-antibody complex from unbound antigen, and is therefore not a satisfactory method from a practical point of view.
In the foregoing situations, it is quite difficult to measure the quantity of hPH precisely in a simple manner by measuring the enzymatic activity. Thus, there is a great demand for developing a new method for effectively and precisely determining hPH in a simple manner in place of the conventional methods accompanied with various disadvantages, especially in the field of diagnosis of hepatic diseases.